Recent Advancements in Diagnosis and Management of Livestock and Poultry Diseases

Recent Advancements in Diagnosis and Management of Livestock and Poultry Diseases

Manoranjan Rout*and Jajati Keshari Mohapatra

ICAR-National Institute on Foot and Mouth Disease, International Centre for Foot and Mouth Disease, Bhubaneswar, Odisha – 752 050, India

*Corresponding email: drmrout@gmail.com

Introduction and Backdrop

Livestock sector contributes 4.9% of the GDP and nearly 28.4% to national agricultural GDP (Annual Report 2019-2020, Ministry of Fisheries, Animal Husbandry and Dairying, 2020). India owns 536.76 million livestock population and 851.81 million poultry birds (20^th Livestock Census2019, All India Report, 2019). The livestock and poultry sector significantly influence the economy of the country.Despite such high number of livestock resources available in the country, its growth in terms of health and productivity gets victimized by different infectious diseases. In order for prevention and control of such diseases, their diagnosis is the base, as diagnosis is an essential element of disease prevention and management. The World Organization of Animal Health (WOAH) in this regard prescribes the application of rapidand highly sensitive techniques for detection of the causative agents so that necessary measures can be implemented to prevent its further spread. In the present paper, the recent advancements in disease diagnosis have been discussed in nutshell.

Significant livestock diseases in India

Global livestock population has long been victimized by infectious diseases caused by various pathogens including bacteria, viruses and parasites. Rapid and precise diagnosis is the base that can give a right direction for an accurate line oftreatment of the infected animals and birds. Barman et al.(2020) have listed out some important prevalent diseases in the country e.g., foot and mouth disease (FMD), porcine reproductive and respiratory syndrome (PRRS), bluetongue (BT), bovine viral diarrhea (BVD),brucellosis, bovine herpes viral infection (BHV), classical swine fever (CSF), porcine circovirus disease (PCV-1 and 2), Peste des petits ruminants (PPR), hemorrhagic septicemia (HS), bovine tuberculosis (TB), leptospirosis, papillomatosis, mastitis, rabies, porcine cysticercosis (PC), etc. African swine fever (ASF) and lumpy skin disease(LSD) can be added further with their reports in the country in near past. In poultry, Marek’s disease (MD), Ranikhet/Newcastle disease (RD or ND), infectious bronchitis (IB), infectious bursal disease (IBD), avian influenza (AI) etc. bear immense significance with attention of the health authority.

Diagnostic platforms/strategies in today’s scenario

The most recent ‘one-health concept’ has compelled the current diagnostic system to follow a multidisciplinary approach for holistic detection of agents. Not the mere diagnosis, but its rapidity and accuracy bear immense significance for the implementation of efficient measures in order tocheck its spread. Despite the advancements in the available diagnostic platforms, a battery of classical and conventional techniques appears to be useful in diagnosis of infectious diseases that has been adding value to the service. To cite a few, among such techniques, serology, cell culture, electron microscopy are useful though being time-consuming or labour-intensive. One of such traditional approach is isolation of agents in sensitive cell culture system that is effective but expensive demanding much time. However, such isolation of agents is considered to be the ‘gold standard’ in diagnostic pathology. Other traditional methods like serology, microscopy, complement fixation test, agar gel immunodiffusion test, hemagglutination assay and hemagglutination inhibition assay, latex agglutination test, fluorescent antibody test, radio-immunoassay, ELISA are of course useful.

But with due course of time new inventions have come up filling the deficiencies/gaps in the field of molecular biology and biotechnology leading to the development of newer and robust techniques. Such recent techniques are discussed here that have been used for diagnosis of diseases of livestock and poultry. DNA-based approaches are becoming more common in the laboratories where the polymerase chain reaction (PCR) has become one of the most dependent techniques that was invented and developed in 1985 by Kary B. Mullis. The conventional PCR has been modified to nested and multiplex PCR in order to increase the sensitivity as well as to target multiple pathogens or different target genes of the same agent with a multitarget approach. However, some of the limitations of PCR including quantification of target nucleic acid were addressed and resolved in 1992 by the development of real-time PCR by Higuchi et al. (1992) that further enhanced the diagnostic platform in terms of the sensitivity and robustness. Some other technologies such as random amplified polymorphic DNA (RAPD), loop-mediated isothermal amplification (LAMP), luminex based assays, nanobiotechnology aiding biosensors have been emerged as efficient novel approaches in diagnosis of important diseases. These technologies although being more rapid, sensitive and specific, demand sophisticated equipment with utmost care in handling that limits their utility in on-site monitoring or pen-side diagnosis of diseases. Immuno-assays like enzyme-linked immunosorbent assay (ELISA) though invented much before, is being used till date offering solid diagnosis of different diseases of livestock and poultry. The use of point-of-care (POC) diagnostics provides rapid diagnostic scope obviating the requirement of dedicated laboratories. Obviously, the POC diagnostics have been used in human medicine, but equivalent degree of popularity has not yet been achieved in veterinary field. It has been reported that only two POC devices from a total of 14 diagnostic kits for 11 animal diseases that have been registered to OIE. POC devices have targeted few animal pathogens of economic significance (FMD, ASF, CSF, PRRS,BT) or agents of zoonotic significance (salmonellosis, brucellosis, Campylobacteriosis or vibriosis, Escherichia coli, avian and swine influenza) (Manessis et al., 2022).

Improved techniques for disease diagnosis

Polymerase chain reaction (PCR) and its variants

PCR with a significant sensitivity and specificity for genome/nucleic acid detection has been one of the most dependent as well as widely used diagnostic tools in disease diagnostics today allowing a precise identification of the etiological agents further helping the genetic characterization. This technique targeting specific gene segment provides a platform for differential diagnosis of similar looking diseases in the field of veterinary science. In veterinary field PCR was first applied for genome detection of BVDV, FMDV, infectious bovine rhinotracheitis (IBR), buffalopox and ephemeral fever virus. Mass-scale diagnosis of avian influenza and Newcastle disease in USA has been possible using PCR (Schmitt and Henderson, 2005).

Reverse transcription-PCR (RT-PCR)

RT-PCR is another version of PCR used for detection of RNA transcripts of RNA viral diseases. As the target is RNA, the viral RNA template is first reverse transcribed to complementary DNA (cDNA) using reverse transcriptase enzyme of different origin like Avian Myeloblastosis Virus Reverse Transcriptase (AMVRT) or Moloney Murine Leukemia Virus Reverse Transcriptase (MMLVRT). The cDNA is further used as a template for PCR amplification. Presently, this is one of the most sensitive techniques available for detection of RNA virus offering a reliable diagnosis of many diseases like FMD, avian influenza, rotavirus, BT virus (Callens and De Clercq, 1999).

Nested PCR

Nested PCR, a modification of the conventional PCR helps minimizing non-specific binding. The first set of primers is designed to bind to the sequences upstream from the second set of primers and is used in an initial PCR reaction. Because of such nested approach, its detection sensitivity has been remarkably enhanced in detection of infectious agents. Nested PCR has been used for the detection of arbovirus, avian leucosis virus (ALV), NDV, FMDV, avian influenza virus, canine corona virus, West Nile virus and orf virus (Pratelli et al., 2004; Bora et al., 2015).

Multiplex PCR (mPCR)

Multiplex PCR has proven to be very useful for rapid and simultaneous identification of multiple pathogens or multiplexing different target genes of the same pathogen in a given sample. This technique has provided platform for the diagnosis of campylobacteriosis, leptospirosis, FMD. Simultaneous detection of CSFV, porcine parvovirus (PPV), PCV-2 and PRRSV in pigs has successfully been possible using mPCR. One such mPCR assay has been developed to detect H5N1 virusand mastitis causing pathogens in animals (Shome et al., 2011).

Real-time PCR

Real-time PCR is the advancement over the conventional PCR allowing the amplification readings on real time basis as well as eliminating the post-PCR processing. The technique offers benefit of target quantification with the use of TaqMan probes, SYBR green dye, scorpion probes, molecular beacons etc. Because of the singletube reaction set up, the probability of cross-contamination between samples is greatly reduced. Detection of slowgrowing intracellular pathogens like Mycobacterium, Histoplasma and Brucella has made use of this technique. It has successfully detected BTV (Maan et al., 2015), FMDV and bovine piroplasmids.

Nucleic acid sequence-based amplification (NASBA) and Transcription mediated amplification (TMA)

NASBA and TMA are similar isothermal amplification techniques that proceed through RNA. The isothermal amplification is dependent upon a constant temperature of 41°C. It involves twostep process where, there is an initial enzymatic amplification of the target nucleic acid followed by detection of the amplified amplicons. Being dependent on a single temperature, it obviates the need of a PCR machine or thermocycler.

Loop-mediated isothermal amplification (LAMP)

LAMP assay, a simple, rapid, specific technique originally described by Notomi et al. (2000) has been an important technique used in the developing countries because of its cost-effectiveness. The assayis performed in a single step and is completed within 1 hour with incubation of samples, primers and Bst DNA polymerase and substrates at a constant temperature of 60-65°C. Being an isothermal amplification technique, it demands no thermocycler without requiring further post-PCR processing of samples. It has been used for detection of important veterinary pathogens e.g., FMDV (Yamazaki et al., 2013), capripox viruses (Batra et al., 2015), BTV (Maan et al., 2016) and PPRV (Zhan et al., 2009).

Random amplified polymorphic DNA (RAPD)

RAPD, a simple, fast and inexpensive technique is a type of PCR, where the segments of DNA amplified are random. Several arbitrary, short primers (10-12 nucleotides) are created followed by PCR using a large template of genomic DNA. In this method, knowledge of the DNA sequence of the target genome is not a pre-requisite. The assay relies on amplification of genomic DNA with a single primer chosen from an arbitrary sequence and has been used for strain description in epidemiological studies. It has problems in experiment reproducibility. The primer and template mismatch may lead to total absence as well as decreased amount of the PCR product.

Amplified fragment length polymorphism (AFLP)

AFLP, a PCR-based tool developed in the early 1990s is used in genetics, DNA fingerprinting and genetic engineering. AFLP is a highly sensitive method for polymorphisms in DNA that was originally described by Zabeau and Vos in 1993. In a nutshell, AFLP is the selective amplification of restriction fragments from a digest of total genomic DNA using PCR. Restriction enzymes are used to digest genomic DNA followed by ligation of adaptors to the sticky ends of the restriction fragments. A subset of the restriction fragments is then selected to be amplified.

Restriction fragment length polymorphism (RFLP)

RFLP exploits variations in homologous DNA sequences known as polymorphisms. Based on the patterns obtained after enzymatic cuttings of their DNA, the method can differentiate the target pathogen. The restriction enzymes digest the DNA sample into fragments and the fragments are subsequently segregated by gel electrophoresis based on their size. The cleavage patterns generated after enzymatic digestion are used to differentiate species and strains from one another. However, RFLP is now largely obsolete due to the emergence of sophisticated DNA sequencing technique, but it was the first DNA profiling technique inexpensive enough to witness widespread application.

Microarray technology

DNA microarray popularly referred to as DNA chip/gene chip, is an advancement in the field of molecular biology being used for the detection of a wide range of pathogens of medical as well as veterinary importance (Salih et al., 2015). This method combines DNA amplification with subsequent hybridization to oligonucleotide probes targeting multiple sequences of interest. Microarray has the ability to differentiate different etiological agents exhibiting similar looking clinical signs e.g., diseases producing vesicular lesions like vesicular stomatitis, vesicular exanthema and swine vesicular disease. Detection of Mycoplasma mycoides subsp mycoides has been done using this technique.

Nanotechnology/nanobiotechnology

Nanotechnology pertains to the study of extremely small (10^-9) structures having size of 0.1 to 100 nm that is being used in nanomedicine for successful and improved diagnosis of disease that could further guide theproper treatment. The extremely small size of the particles has increased the sensitivity of its use in diagnostic nanomedicine. Gold nanoparticles (AuNPs) are nanostructures having utility in different field of biomedical science. Nanotechnology has proven its applicationin development of molecular diagnostics, rapid pen-side kits or chip-based diagnostic aids for use in human and veterinary medicine.

Biosensing technology

Biosensors aidin fast and efficient diagnosis of infectious diseaseswith much convenience satisfying the ‘pen-side’ factor due to their simplicity and miniaturization. Two primary components fulfil the requirements of a biosensor e.g., bioreceptor and transducer. The bioreceptor recognizes the target analyte, while the transducer converts the element of recognition into a measurable signal. Over long past years, an array of biotechnological innovations have led to the development of efficient biosensors for pathogen detection. Novel biosensor technology has been used for diagnosis of many diseases of veterinary significance that are mentioned below:

1. Biosensors have been helpful in detection of many pathogens of veterinary significance e.g., Bacillus anthracis, Streptococcus suis serotype 2, Salmonella sp., Brucella melitensis, Brucella abortus, avian influenza virus, BVDV, fowl adenovirus 9, Babesia bovis, infectious bronchitis virus (IBV), muscovy duck parvovirus, PRRSV, FMDV, vesicular stomatitis virus (VSV) etc. (Manessis et al., 2022).
2. DNA microarrays have been used for development of multiplex biochips for detection of various dairy pathogens targeting genes of mastitis pathogens, mycoplasma biomarkers, bovis as well as six other pathogens in bovine milk (Lee et al., 2008).
3. Electrochemical biosensor based on molecular beacon for detection of bovine respiratory syncytial virus (BRSV) has been reported. Nanoparticle amplification of immuno-PCR assay has detected RSV.
4. A quantum-dot based lateral-flow immunoassay system was proposed for quantitative detection of influenza A virus subtypes H5 and H9.
5. Novel biosensors relying on specific biochemical recognition strategies have been reported for rapid and specific detection of Escherichia coli causing colibacillosis in poultry, cattle, pigs, goats.
6. Researchers have used single-stranded DNA aptamer with high affinity and specificity against P48 protein of bovis in sera.
7. Braiek et al. (2012) developed an electrochemical sensor for detection of pathogenic Staphylococcus aureus.
8. Cell-based biosensors have been used for detection of Clostridium perfringens and its active toxin. Specific oligoprobes immobilized on a multi-pathogen oligonucleotide microarray have been used to detect six toxin-producing sequences of Perfringens.
9. Bluetongue and epizootic haemorrhagic disease (EHD) were detected using nucleic acid-based assays, DNA microarray and next-generation sequencing (Wilson et al., 2015). Ibaraki virus causing EHD has been identified by magnetic modulation and synchronous detection.
10. New multiplex PCR assays have been used for detection of all seven Eimeria species in poultry (Fernandez et al., 2003).
11. A lateral flow device for recovery of FMDV RNA was reported by Fowler et al. (2014). Reid et al. (2001) developed a chromatographic strip for detection of FMDV antigen. RT-LAMP has also been coupled with lateral flow strip for FMD diagnosis (Waters et al., 2014).
12. Barletta et al. (2013) developed a test based on melting-point curve analysis for identification of jejuni. Specific point-of-care (POC) devices have been developed to detect Campylobacter. Magneto strictive particle coated with three layered silica to bind anti-C. Jejuni antibodies have been used by Zhang et al. (2015).
13. Chai et al. (2012) described magneto elastic mass-sensitive biosensor for the detection of typhimurium. A lateral flow strips for Salmonella using colloidal gold conjugated anti-Salmonella antibodies have been usedto detectthe bacteriumin various animal products.

‘Omics’ era revolutionizing the diagnostics platform

‘Omics’ technologies including genomics, transcriptomics, proteomics etc play very important roles in the diagnosis of infectious diseases as well as many other research applications (Cantacessi et al., 2012). Proteomics is the study of proteins present in a tissue or fluid, while transcriptomics deals with the genome-wide identification and quantification of mRNAs, non-coding RNAs and small RNAs in healthy and disease state.

Next generation sequencing (NGS)

Next generation sequencing technology has enabled a precise and massive parallel sequencing without prior knowledge of the complete DNA content in a test sample allowing for targeted sequencing (Van Borm et al., 2015). Different sophisticated high-thoroughput platforms available in these days have made the different aspects of molecular biology and disease diagnosis more convenient and easier providing valuable information.

 Conclusions

Any disease diagnosis in the field situation, apart from observable clinical signs and lesions, molecular diagnostics have paved the way for a solid confirmation of the disease and characterization of the pathogens. Such advanced settings in diagnostics have not only improvised the speed of diagnosis, but also taken extreme care in the accuracy. Because of such accurate and solid diagnosis, disease epidemiology has become more convenient and comprehensive allowing implementation of proper biosecurity measures in the face of outbreaks. Novel advancements like the point of care tests have proven their portability for convenient application in the field to offer a diagnosis at the farmer’s point. The era of ‘omics’ has greatly supported in diverse fields of science and technology. Such technological advancements in the field of disease diagnosis besides helping in the diagnosis of disease and detection of the pathogens have also been able to support the disease management of livestock and poultry acting as a boon for the farming community who solely depend upon them for earning their livelihood.

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Recent Advancements in the Diagnosis and Management of Livestock and Poultry Diseases